Craig Ag

Structure and Biological Activity of Gonadotropin-Releasing Hormone Isoforms Isolated from Rat and Hamster Brains

Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp9]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp9]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp9]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp9]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues.

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His5,Trp7,Leu8]-GnRH, dogfish (df) GnRH; [His5,Asn8]-GnRH, catfish (cf) GnRH; and [His5,Trp7,Tyr8]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro (D)-isomers D-arginine (D-Arg) or D-naphthylalanine (D-Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of D-Nal6 into [His5,Asn8]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of D-Nal6 or D-Arg6 into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [D-Nal6,Pro9NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [D-Nal6,Pro9NEt]-mGnRH (kd = 0.40 +/- 0.04 and 0.55 +/- 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.

The disulfide bond arrangement in the extracellular domain of the activin type II receptor.

The initial step in the signaling cascade of the growth factor activin involves its binding to the extracellular domain of the activin type II receptor. This receptor domain contains 10 cysteine residues which are engaged in intramolecular disulfide bonds. To elucidate the structural framework of this domain we have characterized its disulfide-bonding pattern using an extracellular fragment of the receptor which binds activin A with high affinity. By combining proteolysis with mass spectroscopy and chemical sequence analysis, the disulfide connectivity was determined to be as follows: C1–C3, C2–C4, C5–C8, C6–C7, and C9–C10. A similar disulfide arrangement occurs in a family of snake toxins for which the three-dimensional structure is known.

Competitive antagonists of the corticotropin releasing factor (CRF) scanned with a i-(i+3) Glu Lys Bridge

CRF [1] is involved in a wide spectrum of central nervous system (CNS)-mediated effects, suggesting that this peptide plays an important role within the brain, especially in response to stressful stimuli [2]. Systematic SAR investigations have resulted in the development of CRF antagonists such as [3], members of the (standard) family [4] and conformationally restricted analogs [5] that are effective in the CNS. Those results, predictive methods and physicochemical measurements have suggested that CRF and its family members (urotensins and sauvagine) assume an conformation when interacting with the CRF receptors. To further test this hypothesis, we have scanned the whole rat/human sequence with an i-(i + 3) bridge consisting of the Glu-Xaa-Xbb-Lys scaffold which we and others had shown to be compatible with maintenance or enhancement of structure in at least some unpredictable cases.